Split EPR signals from photosystem II are modified by methanol, reflecting S state-dependent binding and alterations in the magnetic coupling in the CaMn4 cluster

Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.     

Effects of methanol on the S i -state transitions in photosynthetic water-splitting

From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at-or at least very close to-the Mn(4)O(x)Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O(2) evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S( i ) state advancement on the methanol concentrations in the range of 0-10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn(4)O(x)Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S(4) --> S(0) and the other during the S(2) --> S(3) transition.     

Neurophilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells

Photosystem II complex (PSII) of thylakoid membranes uses light energy to oxidise extremely stable water and produce oxygen (2H(2)O-->O(2)+4H(+)+4e(-)). PSII is compared with cytochrome c oxidase that catalyses the opposite reaction coupled to proton translocation. Cytochrome c oxidase has proton and water channels, and a tentative oxygen channel. I propose that functional PSII complexes also need a specific oxygen channel to direct O(2) from the water molecules bound to specific Mn atoms of the Mn cluster within PSII out to the membrane surface. The function of this channel will be to prevent oxygen being accessible to the radical pair P680(+)Pheo(-), thereby preventing singlet oxygen generation from the triplet P680 state in functional PSII. The important role of singlet oxygen in structurally perturbed non-functional photosystem II is also discussed.     

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations

The Mn(4)Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn(4)Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn(4)Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca(2+) and Cl(-) from the active site.     

The Psb27 protein facilitates manganese cluster assembly in photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.     

Detection of an electron paramagnetic resonance signal in the S0 state of the manganese complex of photosystem II from Synechococcus elongatus.

The Mn(4)-cluster of photosystem II (PSII) from Synechococcus elongatus was studied by electron paramagnetic resonance (EPR) spectroscopy after a series of saturating laser flashes given in the presence of either methanol or ethanol. Results were compared to those obtained in similar experiments done on PSII isolated from plants. The flash-dependent changes in amplitude of the EPR multiline signals were virtually identical in all samples. In agreement with earlier work [Messinger, J., Nugent, J. H. A., and Evans, M. C. W. (1997) Biochemistry 36, 11055-11060; Ahrling, K. A., Peterson, S., and Styring, S. (1997) Biochemistry 36, 13148-13152], detection of an EPR multiline signal from the S(0) state in PSII from plants was only possible with methanol present. In PSII from S. elongatus, it is shown that the S(0) state exhibits an EPR multiline signal in the absence of methanol (however, ethanol was present as a solvent for the artificial electron acceptor). The hyperfine lines are better resolved when methanol is present. The S(0) multiline signals detected in plant PSII and in S. elongatus were similar but not identical. Unlike the situation seen in plant PSII, the S(2) state in S. elongatus is not affected by the addition of methanol in that (i) the S(2) multiline EPR signal is not modified by methanol and (ii) the spin state of the S(2) state is affected by infrared light when methanol is present. It is also shown that the magnetic relaxation properties of an oxidized low-spin heme, attributed to cytochrome c(550), vary with the S states. This heme then is in the magnetic environment of the Mn(4) cluster.     

Visible light induction of an electron paramagnetic resonance split signal in Photosystem II in the S(2) state reveals the importance of charges in the oxygen-evolving center during catalysis: a unifying model

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(?), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(?)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.     

The basic properties of the electronic structure of the oxygen-evolving complex of photosystem II are not perturbed by Ca2+ removal

Ca(2+) is an integral component of the Mn(4)O(5)Ca cluster of the oxygen-evolving complex in photosystem II (PS II). Its removal leads to the loss of the water oxidizing functionality. The S(2)' state of the Ca(2+)-depleted cluster from spinach is examined by X- and Q-band EPR and (55)Mn electron nuclear double resonance (ENDOR) spectroscopy. Spectral simulations demonstrate that upon Ca(2+) removal, its electronic structure remains essentially unaltered, i.e. that of a manganese tetramer. No redistribution of the manganese valence states and only minor perturbation of the exchange interactions between the manganese ions were found. Interestingly, the S(2)' state in spinach PS II is very similar to the native S(2) state of Thermosynechococcus elongatus in terms of spin state energies and insensitivity to methanol addition. These results assign the Ca(2+) a functional as opposed to a structural role in water splitting catalysis, such as (i) being essential for efficient proton-coupled electron transfer between Y(Z) and the manganese cluster and/or (ii) providing an initial binding site for substrate water. Additionally, a novel (55)Mn(2+) signal, detected by Q-band pulse EPR and ENDOR, was observed in Ca(2+)-depleted PS II. Mn(2+) titration, monitored by (55)Mn ENDOR, revealed a specific Mn(2+) binding site with a submicromolar K(D). Ca(2+) titration of Mn(2+)-loaded, Ca(2+)-depleted PS II demonstrated that the site is reversibly made accessible to Mn(2+) by Ca(2+) depletion and reconstitution. Mn(2+) is proposed to bind at one of the extrinsic subunits. This process is possibly relevant for the formation of the Mn(4)O(5)Ca cluster during photoassembly and/or D1 repair.     

QM/MM computational studies of substrate water binding to the oxygen-evolving centre of photosystem II

This paper reports computational studies of substrate water binding to the oxygen-evolving centre (OEC) of photosystem II (PSII), completely ligated by amino acid residues, water, hydroxide and chloride. The calculations are based on quantum mechanics/molecular mechanics hybrid models of the OEC of PSII, recently developed in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The model OEC involves a cuboidal Mn3CaO4Mn metal cluster with three closely associated manganese ions linked to a single mu4-oxo-ligated Mn ion, often called the 'dangling manganese'. Two water molecules bound to calcium and the dangling manganese are postulated to be substrate molecules, responsible for dioxygen formation. It is found that the energy barriers for the Mn(4)-bound water agree nicely with those of model complexes. However, the barriers for Ca-bound waters are substantially larger. Water binding is not simply correlated to the formal oxidation states of the metal centres but rather to their corresponding electrostatic potential atomic charges as modulated by charge-transfer interactions. The calculations of structural rearrangements during water exchange provide support for the experimental finding that the exchange rates with bulk 18 O-labelled water should be smaller for water molecules coordinated to calcium than for water molecules attached to the dangling manganese. The models also predict that the S1-->S2 transition should produce opposite effects on the two water-exchange rates.     

No evidence from FTIR difference spectroscopy that aspartate-170 of the D1 polypeptide ligates a manganese ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Near-infrared-induced transitions in the manganese cluster of photosystem II: action spectra for the S2 and S3 redox states

The Mn4Ca complex that is involved in water oxidation in PSII is affected by near-infrared (NIR) light in certain redox states and these phenomena can be monitored by electron paramagnetic resonance (EPR) at low temperature. Here we report the action spectra of the NIR effects in the S2 and S3 states in PSII from plants and the thermophilic cyanobacterium Thermosynechococcus elongatus. The action spectra obtained are very similar in both S states, indicating the presence of the same photoactive form of the Mn4Ca complex in both states. Since the chemical nature of the photoactive species is not known, an unequivocal interpretation of this result cannot be made; however, it appears to be more easily reconciled with the view that the redox state of the Mn4Ca cluster does not change from the S2 to the S3 transition, at least in those centers sensitive to NIR light. The temperature dependence of the NIR effect and the action spectra for S2 indicate the presence of structural heterogeneity in the Mn4Ca cluster.     

Effects of ammonia on the structure of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

NH(3) is a structural analogue of substrate H(2)O and an inhibitor to the water oxidation reaction in photosystem II. To test whether or not NH(3) is able to replace substrate water molecules on the oxygen-evolving complex in photosystem II, we studied the effects of NH(3) on the high-frequency region (3750-3550 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (pH 7.5 at 250 K), where OH stretch modes of weak hydrogen-bonded active water molecules occur. Our results showed that NH(3) did not replace the active water molecule on the oxygen-evolving complex that gave rise to the S(1) mode at ~3586 cm(-1) and the S(2) mode at ~3613 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. In addition, our mid-frequency FTIR results showed a clear difference between pH 6.5 and 7.5 on the concentration dependence of the NH(4)Cl-induced upshift of the S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectra of NH(4)Cl-treated PSII samples. Our results provided strong evidence that NH(3) induced this upshift in the spectra of NH(4)Cl-treated PSII samples at 250 K. Moreover, our low-frequency FTIR results showed that the Mn-O-Mn cluster vibrational mode at 606 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the NaCl control PSII sample was diminished in those samples treated with NH(4)Cl. Our results suggest that NH(3) induced a significant alteration on the core structure of the Mn(4)CaO(5) cluster in PSII. The implication of our findings on the structure of the NH(3)-binding site on the OEC in PSII will be discussed.     

Detection of structural changes upon S1-to-S2 transition in the oxygen-evolving manganese cluster in photosystem II by light-induced Fourier transform infrared difference spectroscopy.

The light-induced Fourier transform infrared (FT-IR) difference spectrum between the S1 and S2 states of the O2-evolving photosystem II (PSII) was obtained for the first time. Detection of an S2/S1 difference spectrum virtually free from contributions by the acceptor-side signals was achieved by employing an exogenous electron acceptor, potassium ferricyanide, to trypsin-treated PSII membranes and using one-flash excitation at 250 K. A synthetic difference spectrum obtained by adding this S2/S1 spectrum to the QA-/QA spectrum measured with Mn-depleted PSII was almost identical with the difference spectrum of the S2QA-/S1QA charge separation measured with untreated PSII. This successful simulation verifies the correctness of the S2/S1 spectrum thus obtained. The observed S2/S1 spectrum reflects the structural changes within the water-oxidizing Mn cluster upon the S1-to-S2 transition, most probably changes in vibrational modes of ligands coordinating to the Mn ion(s) that is (are) oxidized upon the S2 formation and/or changes in protein conformation. The present results demonstrate that FT-IR difference spectroscopy is a promising method to investigate the structure of the intermediates of the Mn cluster involved in photosynthetic water oxidation.     

Identification of a Mn-O-Mn cluster vibrational mode of the oxygen-evolving complex in photosystem II by low-frequency FTIR spectroscopy

We have developed conditions for recording the low-frequency S(2)/S(1) Fourier transform infrared difference spectrum of hydrated PSII samples. By exchanging PSII samples with buffered (18)O water, we found that a positive band at 606 cm(-)(1) in the S(2)/S(1) spectrum in (16)O water is clearly downshifted to 596 cm(-)(1) in (18)O water. By taking double-difference (S(2)/S(1) and (16)O minus (18)O) spectra, we assign the 606 cm(-)(1) mode to an S(2) mode and also identify a corresponding S(1) mode at about 625 cm(-)(1). In addition, by Sr and (44)Ca substitution experiments, we found that the 606 cm(-)(1) mode is upshifted to about 618 cm(-)(1) by Sr(2+) substitution but that this mode is not affected by substitution with the (44)Ca isotope. On the basis of these results and also on the basis of studies of Mn model compounds, we assign the 625 cm(-)(1) mode in the S(1) state and the 606 cm(-)(1) mode in the S(2) state to a Mn-O-Mn cluster vibration of the oxygen-evolving complex (OEC) in PSII. This structure may include additional bridge(s), which could be another oxo, carboxylato(s), or atoms derived from an amino acid side chain. Our results indicate that the bridged oxygen atom shown in this Mn-O-Mn cluster is exchangeable and accessible by water. The downshift in the Mn-O-Mn cluster vibration as manganese is oxidized during the S(1) --> S(2) transition is counterintuitive; we discuss possible origins of this behavior. Our results also indicate that Sr(2+) substitution in PSII causes a small structural perturbation that affects the bond strength of the Mn-O-Mn cluster in the PSII OEC. This suggests that Sr(2+), and by inference, Ca(2+), communicates with, but is not integral to, the manganese core.     

Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn(4)Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn(4)Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H(2)(18)O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S(3) state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S(1) state Mn-EXAFS spectrum, a slightly altered S(2) state multiline EPR signal, a substantially altered S(2)-minus-S(1) FTIR difference spectrum, and an unusually long lifetime for the S(2) state (>10 h) in a substantial fraction of reaction centers. In contrast, the S(2) state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S(2)-minus-S(1) FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with (15)N and specific labeling with l-[1-(13)C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO(-) group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm(-1) in both the S(1) and S(2) states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S(2) state and that most of these can achieve the S(3) state, but no evidence for advancement beyond the S(3) state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn(4)Ca cluster in the S(2) state, the FTIR and H(2)(18)O exchange data show that the mutation strongly influences other properties of the Mn(4)Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S(1) to S(2) transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S(3) state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn(4)Ca cluster in the S(1) state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S(1) to S(2) transition. The H(2)(18)O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S(3) state.     

Mono-manganese mechanism of the photosystem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S(0) through S(4), to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn(4)Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The (18)O exchange rates of two substrate water molecules were recently measured for four S(i)-state samples (i=0-3) leading to (34)O(2) and (36)O(2) formations, revealing asymmetric substrate binding sites significantly depending on the S(i)-state. In this paper, we present a chemically complete model for the Mn(4)Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 A resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of (18)O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (mu-oxo, mu-carboxylato, and a hydrogen bond) to the Mn(3)CaO(3)(OH) core, by developing a generalized theory of (18)O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl(-) ion, may be bound to CP43-Arg357 nearby Ca(2+) ion and that D1-His337 may be used to trap a released proton only in the S(2)-state.     

The S0 state of photosystem II induced by hydroxylamine: differences between the structure of the manganese complex in the S0 and S1 states determined by X-ray absorption spectroscopy

Hydroxylamine at low concentrations causes a two-flash delay in the first maximum flash yield of oxygen evolved from spinach photosystem II (PSII) subchloroplast membranes that have been excited by a series of saturating flashes of light. Untreated PSII membrane preparations exhibit a multiline EPR signal assigned to a manganese cluster and associated with the S2 state when illuminated at 195 K, or at 273 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). We used the extent of suppression of the multiline EPR signal observed in samples illuminated at 195 K to determine the fraction of PSII reaction centers set back to a hydroxylamine-induced S0-like state, which we designate S0*. The manganese K-edge X-ray absorption edges for dark-adapted PSII preparations with or without hydroxylamine are virtually identical. This indicates that, despite its high binding affinity to the oxygen-evolving complex (OEC) in the dark, hydroxylamine does not reduce chemically the manganese cluster within the OEC in the dark. After a single turnover of PSII, a shift to lower energy is observed in the inflection of the Mn K-edge of the manganese cluster. We conclude that, in the presence of hydroxylamine, illumination causes a reduction of the OEC, resulting in a state resembling S0. This lower Mn K-edge energy of S0*, relative to the edge of S1, implies the storage and stabilization of an oxidative equivalent within the manganese cluster during the S0----S1 state transition. An analysis of the extended X-ray absorption fine structure (EXAFS) of the S0* state indicates that a significant structural rearrangement occurs between the S0* and S1 states. The X-ray absorption edge position and the structure of the manganese cluster in the S0* state are indicative of a heterogeneous mixture of formal valences of manganese including one Mn(II) which is not present in the S1 state.     

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.